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Prescreening of Genetic Diseases

(It’s worth & potential)

Dr Pupak Derakhshandeh, PhD

Ass Prof of Medical Science of Tehran University

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Prescreening for• Down syndrome and trisomy 13 & 18• Breast cancer (BRCA1 AND BRCA2 GENES)• Colorectal cancer• SMA carrier testing• Factor V Leiden• Cardiovascular risk with C-reactive protein• and Apolipoprotein E

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Prescreening for Down syndrome and trisomy

21, 13 & 18

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Down Syndrome (Trisomy 21(

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Down SyndromeDown Syndrome (Trisomy 21 (Trisomy 21((

Trisomy 2Trisomy 2((

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First trimester screening for Down syndrome and trisomy 13 & 18

(up to12W) • The availability and acceptability of early

invasive diagnostic methods (eg, chorionic villus sampling, CVS)

• The continued need for second trimester screening for open fetal neural tube defects

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Women with singleton pregnancies: first-trimester combined screening

–measurement of Nuchal translucency

–pregnancy-associated plasma protein A [PAPP-A]

–The free beta subunit of human chorionic gonadotropin (HCG) at 10 weeks 3 days through 13 weeks 6 days of gestation

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Nuchal translucency screening involves the measurement by ultrasound of the skin

thickness at the back of the neck of a first

trimester fetus

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Identification about 85-90% of affected fetuses in the first-trimester

• maternal age was combined with fetal NT

• and maternal serum biochemistry (free β-HCG and pregnancy-associated plasma protein (PAPP-A)

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Second-trimester (13-24W) quadruple screening

–measurement of:

• alpha-fetoprotein

• total human chorionic gonadotropin (HCG)

• unconjugated estriol

• inhibin A at 15 through 18 weeks of gestation

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Maternal serum alpha-fetoprotein (MSAFP)

• fetus has two major blood proteins:

• albumin and alpha-fetoprotein (AFP)

• Since adults typically have only albumin in their blood

• the MSAFP test can be utilized to determine the levels of AFP from the fetus

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MSAFP

• the gestational age must be known with certainty

• the amount of MSAFP increases with gestational age

• Neural tube defect

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Neural tube defect (NTD)

• in the fetus:

• from failure of part of the embryologic neural tube to close

• there is a means for escape of more AFP into the amniotic fluid !

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Note!

• the MSAFP can be elevated for a variety of reasons

• which are not related to fetal neural tube or abdominal wall defects, so this test is not 100% specific

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Neural tube defect

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Maternal blood sampling for fetal blood cells

• This is a new technique

• use of the phenomenon of fetal blood cells gaining access to maternal circulation through the placental villi

• only a very small number of fetal cells enter the maternal circulation in this fashion

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Prenatal screening and diagnosis of neural tube defects

• Neural tube defects (NTD): second most prevalent congenital anomaly in the United States

• Two factors have played a significant role in the prevention of this disorder in developed countries: – Sonographic imaging combined with

amniocentesis for diagnosis of affected fetuses – folic acid supplements for prevention of the

disorder

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Anencephaly (failure of closure at the cranial end of the neural tube)

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Spina bifida (failure of closure at the caudal end of the neural tube)

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Environmental factors• The frequency of NTDs is increased with

exposure to certain environmental factors:– drugs (valproic acid, carbamazepine, Folic acid

deficiency)– diabetes mellitus– Obesity

• Adequate folate is critical for cell division due to its essential role in the synthesis of:– nucleic– certain amino acids

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Genetic factorsthe observations that NTDs have a high rate:

– in monozygotic twins– more frequent among first degree relatives– more common in females than males

• The risk of recurrence for NTDs: approximately 2

to 4 percent when there is one affected sibling

• With two affected siblings, the risk is approximately: 10 percent

• to be higher in countries such as Ireland

where the prevalence if NTDs is high

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NOTE:• The genetic polymorphisms :

• mutations in the methylene tetrahydrofolate reductase gene

• may increase the risk for NTDs

• Folate is a cofactor for this enzyme

• which is part of the pathway of homocysteine metabolism in cells

• The C677T and the A1298C mutations are associated with elevated maternal homocysteine concentrations and an increased risk for NTDs in fetuses

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Prevention of neural tube defects

• can be accomplished by supplementation of the maternal diet with only 4 mg of folic acid per day

• but this vitamin supplement must be taken a month before conception and through the first trimester

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Maternal serum beta-HCG

• the beta-HCG can be used in conjunction with the MSAFP to screen for chromosomal abnormalities, and Down syndrome in particular

• An elevated beta-HCG coupled with a decreased MSAFP suggests Down syndrome

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Maternal serum estriol

• made by the fetal adrenal glands

• Estriol tends to be lower when Down syndrome is present

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Inhibin-A

• An increased level of inhibin-A is associated with an increased risk for trisomy 21

• A high inhibin-A may be associated with a risk for preterm delivery

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Trisomy 21

MSAFP / beta-HCG

estriol / inhibin-A

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overlapping are typical for trisomy 18

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Prescreening BRCA1 AND BRCA2 GENES

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BRCA1 AND BRCA2 GENES

• Breast cancer develops in about 12 percent of women who live to age 90

• a positive family history is reported by 15 to 20 percent of women with breast cancer

• They are associated with an inherited gene mutation

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• Two major susceptibility genes for breast cancer, BRCA1 and BRCA2

• Testing for mutations in these genes, is available

• Clinicians and patients must decide when it is appropriate to screen for their presence

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BRCA mutations• The reason why BRCA mutations

predispose mainly to breast and ovarian cancers is unclear

• intact BRCA1 represents a barrier to transcriptional activation of the estrogen receptor

• that functional inactivation could lead to altered hormonal regulation of mammary and ovarian epithelial proliferation

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BRCA1 or / and BRCA2 gene abnormalities

• Cancer risk with a high penetrance

• women who have inherited mutations

• the lifetime risk of breast cancer is between 65 and 85 percent by age 70

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Ovarian cancer

• Ovarian cancer is also linked to the presence of BRCA mutations

• the lifetime risk of ovarian cancer: – between 45 and 50 percent in women who

have a deleterious BRCA1 mutation– and 15 to 25 percent for those with a BRCA2

mutation

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BRCA2-associated cancers

• prostate cancer• male breast cancer• pancreatic cancer • Although the risk of male breast cancer

and pancreatic cancer may be under 10 percent

• the risk of prostate cancer in BRCA2 carriers may be as high as 35 to 40 percent

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BRCA 1

• The gene Locus for BRCA1: 17q21• a large gene• 24 exons • encoding a 220 kD • 1863 amino acids• Two recognizable motifs

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BRCA2

• BRCA2 (13q12.3)

• was identified by Wooster et al. in 1995

• It encodes for 384 kD nuclear protein

• 3418 amino acids

• BRCA2 bears no homology to any known tumour supressor genes

• contains 27 exons

• spread over 70 kb of genomic DNA

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BRCA1 Gene

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BRCA2 Gene

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SSCP

single strand conformation polymorphism

simplicity clearly by heteroduplex analysis (HA)

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Pedigree of a selected family with breast cancer

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SSCP AnalysisBRCA1 Exon 15, 4650delCA

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Pedigree of a selected family with breast cancer

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SSCP Analysis

BRCA1, Exon 20,Nt 5382

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SSCP AnalysisExon 11pi BRCA1 MS R1347G

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Breast Cancer Families

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Significance of family history

• Degree of relatedness to affected relatives

• Number of affected relatives

• The age of the relative (s) when breast cancer occurred

• Whether there is a family history of ovarian cancer

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Mutations in BRCA1/2 gene

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Prescreening for colorectal cancer

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Screening for colorectal cancer

• Colorectal cancer (CRC) is – common– Lethal– preventable disease (98%)

• It is infrequent before age 40

• the incidence rises progressively to 3.7/1000 per year by age 80

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Clinical detection of increased risk

• Before deciding how to screen:• clinicians should decide whether the individual patient is at

average or increased risk • based on his or her medical and family history• A few simple questions are all that is necessary:• Have you ever had colorectal cancer or an adenomatous polyp• Have you had inflammatory bowel disease (Crohn disease)• Has a family member had colorectal cancer or an adenomatous

polyp• If so, how many• was it a first-degree relative (parent, sibling, or child)• and at what age was the cancer or polyp first diagnosed

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Crohn’s disease

• an inflammatory bowel disease• causes inflammation of the

gastrointestinal tract in both men and women

• persistent diarrhea, abdominal pain, fever, and at times rectal bleeding

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Crohn’s disease

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screening for Colorectal cancer (CRC)

• Patients at highest risk with familial syndromes (HNPCC, FAP)

• should be screened for CRC with colonoscopy at frequent specified intervals

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People at high risk

• a first-degree relative with colon cancer• or adenomatous polyp diagnosed at age

<60 years• or two first-degree relatives diagnosed at

any age• should be advised to have screening

colonoscopy starting at age 40 years• or 10 years younger than the earliest

diagnosis in their family• whichever comes first, and repeated every

five years

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HNPCC

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Microsatellite Instability (MIN) in Adenomas as a Marker for Hereditary

Nonpolyposis Colorectal Cancer

•Hereditary nonpolyposis colorectal cancer (HNPCC)

•the most common of the well-defined colorectal cancer syndromes

•HNPCC: accounting for at least 2% of the total colorectal cancer

•carrying a greater than 80% lifetime risk of cancer

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Microsatellite instability (MIN)

• can be detected in approximately 90% of tumors from individuals with Hereditary Non-Polyposis Colorectal Cancer (HNPCC)

• MIN is also reported in approximately 15% of sporadic colorectal carcinomas

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Mutations in the human mismatch repair genes

(MMR MLH1, MSH2, MSH6, PMS1, PMS2)

• responsible for the MIN of the HNPCC tumors

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Reduction in cancer morbidity and mortality of HNPCC patients

• can be accomplished by appropriate clinical cancer screening of HNPCC patients with mutations in mismatch repair (MMR) genes

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Germline mutation analysis

• In individuals with cancer

• mutation detection can be accomplished relatively efficiently by germline mutation analysis of individuals (blood) whose cancers show microsatellite instability (MIN)

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• Among 378 adenoma patients• six (1.6%) had at least one MIN adenoma• Five out of the six patients (83%) had a

germline MMR gene (mismatch repair gene) mutation

• MIN analysis is a useful method of MIN analysis is a useful method of prescreening colorectal adenoma patients prescreening colorectal adenoma patients for HNPCCfor HNPCC

Microsatellite Instability in Adenomas as a Marker for Hereditary Nonpolyposis Colorectal Cancer Anu Loukola et al. American Journal of Pathology. 1999;155:1849-1853